Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca(2+)-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca(2+) ([Ca(2+)]i) via Ca(2+) influx, Ca(2+) mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca(2+) via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca(2+)]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca(2+) concentration in the ER, a compensatory up-regulation of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and a reduction in depolarization-evoked Ca(2+) influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.
Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Membrane Permeability ; drug effects ; Cells, Cultured ; Down-Regulation ; drug effects ; Endoplasmic Reticulum ; metabolism ; Glucose ; pharmacology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; Potassium Chloride ; pharmacology ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism ; Thapsigargin ; pharmacology ; Up-Regulation ; drug effects

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an alpha-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.
Amino Acid Sequence ; Animals ; Antigens/genetics/*metabolism ; Base Sequence ; Blotting, Western ; Chromatography, Liquid ; *Cloning, Molecular ; DNA, Complementary/genetics ; Epitopes ; Gene Expression Regulation/*physiology ; Ixodidae/genetics/*metabolism ; Molecular Sequence Data ; Tandem Mass Spectrometry

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.
Animals ; Antigens/chemistry/genetics/*immunology/isolation & purification ; Arthropod Proteins/chemistry/genetics/*immunology/isolation & purification ; Chromatography, Affinity ; Cloning, Molecular ; Cluster Analysis ; Conserved Sequence ; Dermacentor/*genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/genetics ; Gene Expression ; Humans ; Molecular Sequence Data ; Molecular Weight ; Phylogeny ; Recombinant Proteins/chemistry/genetics/immunology/isolation & purification ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

Objective:To investigate the clinicopathological features, immunohistochemical phenotypes and molecular characteristics of adenosquamous carcinoma of the lung(ASC)in elderly patients.Methods:Clinical data of 72 ASC patients in the Department of Pathology, The Second Affiliated Hospital of Soochow University from January 2009 to December 2020 were retrospectively analyzed, and 48 patients aged ≥60 years were selected.Clinical manifestations, imaging findings, histopathological and immunohistochemical characteristics were collected, and gene mutations were detected by the amplification refractory mutation system(ARMS-PCR).Results:There were 48 patients including 32 males and 16 females with a mean age of 70 years(range: 60-84 years). The maximum diameters of the tumors ranged from 0.3 to 9.0 cm(mean: 2.8 cm). Microscopically, the tumors contained two components, squamous cell carcinoma and adenocarcinoma, with the squamous cell carcinoma tissue showing intercellular bridges and the adenocarcinoma tissue showing papillary, acinar or tubular structures.Immunohistochemistry assays detected varying expression levels of CK7(30/31), CK5/6(20/28), TTF1(12/31), P40(15/17), and P63(12/13). Molecular testing showed that the EGFR mutation rate was 58.8%(10/17)and the ALK fusion mutation rate was 5.9%(1/17), while ROS1 and MET mutations were not detected.All 48 patients underwent surgical resection.Conclusions:ASC cases are relatively rare and prone to misdiagnosis.The diagnosis requires the combination of HE morphology, immunohistochemistry and imaging examination, and surgery is the main treatment option.The mutation rate of the EGFR gene is relatively high in ASC patients.

Objective To explore the application effect of new improved 3D printing individualized guidance(3D psi)in total knee arthroplasty(TKA)for knee osteoarthritis(KOA).Methods A total of 100 patients with KOA in 920th Hospital of Joint Logistics Support Force,PLA from January 2021 to January 2022 were selected,and were divided into 2 groups of 50 patients each using the randomized numerical table method.The control group was treated with conventional TKA,and the study group was treated with new improved 3D psi assisted TKA.The operation conditions,postoperative rehabilitation,complications,prosthesis component position deviation,knee range of motion(ROM),lower limb force line parameters[coronal distal femoral mechanical axis lateral angle(mldfa),lower limb mechanical axis angle(HKA)],gait parameters(percentage of support time,stride,pace),knee function(HSS score),quality of life(AIMS2 score)were observed.Results Com-pared with control group,the amount of intraoperative and postoperative blood loss and drainage volume 2 days after operation were less in the study group,and the operation time and hospital stay were shorter(P<0.05).The deviations of LTC Angle,FFC Angle,HKA Angle,LFC Angle and FTC Angle in the study group were smaller than those in the control group(P<0.05).At 3 months,6 months and 12 months after surgery,the percentage of knee ROM,supporting time,stride length and walking speed of the research group were higher than those of the control group,while the coronal-position mLDFA and HKA were lower than those of the control group(P<0.05).The proportion of WBC and PMN in joint fluid at 3 months,6 months and 12 months after surgery was lower than that in control group(P<0.05).The HSS score of the study group was higher than that of the control group at 3 months,6 months and 12 months after operation,and the AIMS2 score was lower than that of the control group(P<0.05).There was no statistically significant difference in the incidence of complications between the study group and the control group(P>0.05).Conclusion The new improved 3D PSI-assisted TKA treatment of KOA can optimize the surgical situation,improve operating accuracy,improve the patient's lower limb alignment,promote limb function recovery,help improve the quality of life,and has high safety.

This study investigated the development characteristics of Dermacentor everestianus under laboratory conditions. The time taken for D. everestianus to complete the whole life cycle was 110.2 days on average, and the average developmental durations of larvae and nymphs were 17.1 days and 29.5 days, respectively. The summation of the prefeeding, feeding, and preoviposition periods of females was 17.8 days, and the oviposition and egg incubation lasted for 18.1 days and 27.7 days, respectively. A highly positive correlation was observed between the weight of engorged female and the number of egg mass laid (r=0.947). The reproductive efficiency index and the reproductive fitness index were 7.1 and 6.1, respectively.
Dermacentor* ; Female ; Genetic Fitness ; Humans ; Larva ; Life Cycle Stages* ; Nymph ; Oviposition ; Ovum

The ticks feed large amount of blood from their hosts and transmit pathogens to the victims. The salivary gland plays an important role in the blood feeding. When the female ticks are near engorgement, the salivary gland gradually loses its functions and begins to rapidly degenerate. In this study, data-independent acquisition quantitative proteomics was used to study changes in the phosphorylation modification of proteins during salivary gland degeneration in Haemaphysalis longicornis. In this quantitative study, 400 phosphorylated proteins and 850 phosphorylation modification sites were identified. Trough RNA interference experiments, we found that among the proteins with changes in phosphorylation, apoptosis-promoting Hippo protein played a role in salivary gland degeneration.

OBJECTIVE To investigate the inhibitory effect of berberine （BER） on the invasion and migration of human renal carcinoma cells and its potential mechanism. METHODS Using human renal carcinoma OSRC-2 cell as object， alamarBlue assay was adopted to detect the inhibitory effects of 0 （control group）， 25， 50， 75， 100， 125， 150， 175 and 200 μmol/L BER on the proliferation of OSRC-2 cell after treatment for 24 h and 48 h. After treated with 0（control group）， 50， 100 μmol/L BER for 48 h， the effect of BER on cell cycle was analyzed by flow cytometry. The migration of OSRC-2 cells in 24 h and 36 h was observed by cell scratch test， and the invasion ability of OSRC-2 cells in 24 h was detected by Transwell assay. The protein expression of methyltransferase-like 3 （METTL3） was detected by Western blot after treatment for 48 h， and RNA methylation quantification kit was used to detect the levels of N6-methyladenosine （m6A） in OSRC-2 cells. RESULTS Compared with control group， BER at different concentrations could significantly decrease the survival rate of OSRC-2 cells （P＜0.01）， and showed a dose-dependent and time-dependent manner. After 48 h of BER treatment at 50， 100 μmol/L， the cell was arrested in G0/G1 phase （P＜0.01）. Compared with control group， the migration and invasion abilities of cells in 50， 100 μmol/L BER group were significantly decreased （P＜0.05 or P＜0.01）； the protein expression of METTL3 and the level of m6A in RNA were significantly decreased （P＜0.01）. CONCLUSIONS BER can inhibit level of m6A by down-regulating the expression of METTL3， thereby inhibiting the invasion and metastasis of human renal carcinoma cells.